P628: HIGH THROUGHPUT IMMUNOGENETIC EVIDENCE FOR EXTENSIVE IN VIVO CLASS SWITCH RECOMBINATION EVENTS IN CLL
Authors: E. Sofou, A. Agathangelidis, A.-M. Antoniadou, E. Georgiou, M. Papaioannou, N. Pechlivanis, F. Psomopoulos, K. Stamatopoulos, A. Chatzidimitriou
Journal: HemaSphere (2022)

Background:In vivo Class Switch recombination (CSR) has previously been reported for a fraction of patients with chronic lymphocytic leukemia (CLL) expressing unmutated IGHV genes (U-CLL) and high mRNA levels of activation-induced cytidine deaminase compared to patients expressing mutated IGHV genes (M-CLL). This observation is certainly intriguing, however inherently limited due to the low-sensitive, Sanger-based approach used for the immunogenetic characterization. Aims: Here, we sought to overcome the aforementioned limitation and obtain more insight into in vivo CSR in CLL using a highly-sensitive, next generation sequencing (NGS) methodology. Methods: We studied 73 patients (48 U-CLL, 25 M-CLL) sampled at diagnosis. Case selection was based on the dominant expression of the IgM isotype, as determined by flow cytometry. IGHV-IGHD-IGHJ gene rearrangements were RT-PCR amplified for the μ, γ, and α transcripts utilizing IGHV Leader and IGHC-specific primers. All obtained PCR products (n=215) were subjected to paired-end NGS. High-quality, productive IGHV-IGHD-IGHJ gene rearrangements (n=23,217,561; median= 97,932/sample) were annotated with IMGT/HighV-QUEST. Clonotypes were defined as rearrangement sequences expressing the same IGHV gene and identical complementarity-determining region 3 (VH CDR3) amino-acid (AA) sequence. Clonotype variants were defined as different nucleotide sequences clustered in the same clonotype. Results: The IGH gene repertoire of μ transcripts was clearly monoclonal in all cases, showing a dominant CLL clonotype (DCC) with a median frequency of 93.5% for either U-CLL or M-CLL. Subsequently, the IGH gene repertoires of γ and α transcripts were scanned for the presence of the DCC. In U-CLL, 46/48 cases expressed switched transcripts with median frequencies of 14% (range 0.01-97.2%) and 21.3% (range 0.1-95.3%) for γ and α transcripts, respectively. In M-CLL, 24/25 patients expressed switched transcripts albeit at significantly lower levels than U-CLL (p=0.01) (median frequencies: 0.2% and 13.6%; ranges 0.03-94% and 0.04-91.2% for the γ and α transcripts, respectively). Interestingly, within U-CLL, cases belonging to stereotyped subset #1 (n=21) and its satellite subset #99 (n=2) exhibited significantly higher levels of switched transcripts (median frequencies: 37.9% and 37.6% for the γ and α transcripts, respectively) than all other cases (p=0.04 for γ transcripts, p=0.002 for α transcripts). Analysis of somatic hypermutation (SHM) in the DCC within the repertoires of μ, γ and α transcripts of U-CLL cases revealed the presence of shared SHMs (even among cases with 100% IGHV gene identity) but also distinct SHMs shared by the γ and α clonotypic transcripts but not the IgM, indicative of common selection pressure post-CSR. Interestingly, M-CLL patients displayed less intraclonal diversity in the γ and α clonotypic transcripts, despite the significant burden of SHM within the IgM DCC. Summary/Conclusion: In conclusion, our data reveal that the B cell receptor IGH gene repertoire is highly dynamic and interconnected in both U-CLL and M-CLL, albeit considerably more pronounced in the former. This is especially the case for patients assigned to the clinically aggressive stereotyped subsets #1 and #99, prompting speculations regarding the precise nature of the respective affinity maturation process. Shared SHMs between clonotype variants only among clonotypic γ and α transcripts rather than the μ transcripts indicate that CSR likely precedes SHM, particularly in U-CLL.